Mitochondria are essential organelles for most eukaryotic cells. They provide diverse metabolic functions including the production of energy and specialized metabolites for biosynthetic processes. To assemble functional mitochondria, proteins and RNAs encoded by both the mitochondrial and nuclear genomes are required. In humans, at least 1,000 nuclear encoded proteins are imported into mitochondria, whereas only 13 proteins of the electron transport chain are encoded by mitochondrial genes. The translation of these few but essential mitochondrial encoded proteins requires mitochondrial ribosomes, translation factors, and transfer RNAs, a subset of which must be imported from the cytosol into the mitochondrial matrix. Recent studies in evolutionarily diverse organisms have shown that multiple RNAs are encoded within the nucleus and imported from the cytosol into mitochondria. Our overall understanding of mitochondrial import pathways for nuclear encoded proteins is quite detailed. By contrast, very little is known about the mitochondrial import mechanism for nuclear encoded RNAs or how this new knowledge can be used to deliver specific RNAs into mitochondria. The revised overall goals of our competitive R01 renewal application are to dissect the main and perhaps only pathway of RNA import into mitochondria and to develop strategies that target nuclear encoded RNAs to mitochondria in reporter constructs and for the treatment of diseases caused by mutations of mitochondrial DNA (mtDNA). In separate studies, we will identify the cohort of RNAs that are imported into mitochondria using next generation RNA-Seq and coupled bioinformatics approaches. Previously, we reported studies that showed that the RNA processing enzyme polynucleotide phosphorylase (PNPase) is unexpectedly localized to the mitochondrial intermembrane space (IMS), where it functions as a gatekeeper for nuclear encoded RNAs that are imported from the cytosol. Interestingly, PNPase is only present in the genomes of organisms such as flies, worms, and mammals, suggesting that its functions are limited to higher eukaryotes. To accomplish our revised proposal goals that retain the original essence of the project, we have identified two specific study aims. In Aim 1, additional regulatory components beyond PNPase for the import of nuclear encoded RNAs will be identified. In Aim 2, reporter and corrective nuclear encoded RNA constructs will be engineered to determine the RNA sequence and structural requirements of the RNA import machinery and to develop strategies to treat diseases caused by mutations in mtDNA. For these studies we will take advantage of different model systems that we have established to manipulate PNPase levels and activities. In addition to increasing our fundamental knowledge about the mechanisms of RNA import into mitochondria and learning the rules for manipulating this RNA import pathway, our renewal application may have a broad impact on public health because our approach could provide a method to ameliorate or possibly cure dozens of human diseases caused by mtDNA mutations.